The Controversial Denouement of Vertebrate DNA Methylation Research

M. Ehrlich

Received November 15, 2004
The study of the biological role of DNA methylation in vertebrates has
involved considerable controversy. Research in this area has proceeded
well despite the complexity of the subject and the difficulties in
establishing biological roles, some of which are summarized in this
review. Now there is justifiably much more interest in DNA methylation
than previously, and many more laboratories are engaged in this
research. The results of numerous studies indicate that some
tissue-specific differences in vertebrate DNA methylation help maintain
patterns of gene expression or are involved in fine-tuning or
establishing expression patterns. Therefore, vertebrate DNA methylation
cannot just be assigned a role in silencing transposable elements and
foreign DNA sequences, as has been suggested. DNA methylation is
clearly implicated in modulating X chromosome inactivation and in
establishing genetic imprinting. Also, hypermethylation of CpG-rich
promoters of tumor suppressor genes in cancer has a critical role in
downregulating expression of these genes and thus participating in
carcinogenesis. The complex nature of DNA methylation patterns extends
to carcinogenesis because global DNA hypomethylation is found in the
same cancers displaying hypermethylation elsewhere in the genome. A
wide variety of cancers display both DNA hypomethylation and
hypermethylation, and either of these types of changes can be
significantly associated with tumor progression. These findings and the
independence of cancer-linked DNA hypomethylation from cancer-linked
hypermethylation strongly implicate DNA hypomethylation, as well as
hypermethylation, in promoting carcinogenesis. Furthermore, various DNA
demethylation methodologies have been shown to increase the formation
of certain types of cancers in animals, and paradoxically, DNA
hypermethylation can cause carcinogenesis in other model systems.
Therefore, there is a need for caution in the current use of
demethylating agents as anti-cancer drugs. Nonetheless, DNA
demethylation therapy clearly may be very useful in cases where better
alternatives do not exist.
KEY WORDS: DNA methylation, cancer, vertebrates

Despite the ubiquitous nature of vertebrate DNA methylation, the history
of research in this field has involved considerable controversy about
its functionality. The first descriptions of 5-methylcytosine
(m5C) in eukaryotic DNA were by Hotchkiss in 1948 [1] and Wyatt in 1951 [2]. Some of
the early research on the species-specific and cancer-specific
distribution of DNA methylation in vertebrate tissues was pioneered by
Boris Vanyushin and colleagues [3-6]. Our laboratory subsequently confirmed the
tissue-specificity of genomic m5C levels in animals [7] and demonstrated such tissue-specificity also for
human specimens [8]. Given the idea that cancer
represents a special kind of derangement of differentiation, we
subsequently looked for and found cancer-specific differences in global
DNA methylation in human tissues [9], as described
below.

In 1975, critical reviews of vertebrate DNA methylation by Holliday and
Pugh [10] and Riggs [11]
advanced our understanding of vertebrate DNA methylation with their
hypotheses about maintenance vs. de novo methylation and the
involvement of this methylation in differentiation and X chromosome
inactivation. In the late 70's and early 80's, there was an initial
flurry of activity to look for associations of differential promoter or
gene methylation with tissue-specific repression or stages of virus
infection [12-16]. Many such
associations were found although many other tissue-specific differences
in DNA methylation did not correlate with expression. Even more genes,
especially constitutively expressed ones, were found to always have
little or no methylation in their promoter regions in a largely
tissue-independent manner. This complexity and the laborious methods
for determining exact patterns of methylation discouraged many
investigators from continuing research in this area. For a while, the
result was insufficient attention to DNA methylation. The main
exception was a small nucleus of scientists from geographically
disperse countries who concentrated their research on the fifth letter
in the vertebrate genomic alphabet.

Another of the impediments to research on vertebrate DNA methylation was
the oversimplified notion emphasized by Walter Gilbert in a 1985
conference [17] that methylation of the vertebrate
genome is probably of little consequence to vertebrate development
simply because Drosophila had not been proven to have DNA
methylation. The reasoning was that Drosophila, like
vertebrates, is a higher eucaryote with complicated developmental
pathways, so if it could accomplish all that differentiation without
DNA methylation, how can vertebrate development use DNA methylation as
an important gene regulator? In the 1980's, this was an often-quoted
idea despite the fact that early Drosophila embryos with their
syncytial development are dramatically different from early vertebrate
embryos and have a much smaller genome. Furthermore, it was already
clear in the 1970's that despite the many common themes in molecular
biology among diverse organisms, considerably different genetic
pathways can yield similar biochemical outcomes. For example, very many
bacterial strains use dam methylation (at the N6
position of the A in GATC) to direct DNA mismatch repair as well as to
regulate gene expression and the initiation of DNA replication [18, 19], but most bacterial
strains do not have dam methylation [20, 21]. These dam methylation-negative bacteria
can use asymmetrical nicks in the DNA generated during discontinuous
DNA replication of one strand to direct mismatch repair [22, 23]. Even the premise that
Drosophila had no genomic m5C was disproved. Recently
it was clearly demonstrated that Drosophila has small amounts of
this methylated base in its genome although this methylation is not
essential for differentiation [24-26], unlike the case for vertebrates (see below).

Vertebrate DNA methylation at transcription control regions appears to
often modulate gene expression or help maintain an already established
inactive state, rather than simply acting as an on-off switch. However,
most methylation of vertebrate genomes is not in such transcription
control elements [5, 7], and
methylation of these elements does not always control gene expression
in vivo [12-16].
Moreover, the inverse correlations between expression and methylation
that are seen for many gene regulatory regions [27-29] could be consequences of
changes in gene expression rather than regulators of such changes.
Among the most convincing examples of changes in DNA methylation
causally involved in initiating transcription control are studies of
imprinted genes (see below).

One of the controversial subjects in this field is whether vertebrate
DNA methylation has mainly a protective role in limiting expression of
foreign DNA elements and endogenous transposons [30] or also is important in the regulation of the
expression of diverse vertebrate genes involved in differentiation [27]. Studies that implicate DNA methylation in
establishing tissue-specific gene expression patterns are often done
with cultured cells. While extensive in vitro methylation of
normally unmethylated promoter regions almost invariably leads to
inhibition of gene expression, there is a need to mimic the same
methylation pattern seen in vivo. This exacting requirement was
accomplished in some studies [31].

Another model system for studying DNA methylation are transgenic mice
with knockout of one of the DNA methyltransferase genes or hypomorphic
alleles of these genes as well as embryonic fibroblasts from these mice
embryos [32-40]. Complicating
studies of the transgenic mice are the multiple activities of all
studied vertebrate DNA methyltransferases [41-48]. This precludes conclusions about the
functionality of vertebrate DNA methylation just from gene knockout
experiments. However, other gene knockouts also globally affect DNA
methylation [49, 50], and
results from those transgenic animals can be used to complement those
from the DNA methyltransferase mutants.

Another important tool for studies of the functionality of DNA
methylation is the use of the methylation inhibitors 5-azacytidine or
the more specific 5-azadeoxycytidine [51, 52]. A caveat for studies with these inhibitors is
that their incorporation into DNA leads to DNA-protein cross-links,
inhibition of DNA replication, and mutation [53,
54], as well as to DNA hypomethylation. Despite
these reservations about individual experimental approaches, an
overview of various kinds of studies convincingly demonstrates the
biological importance of vertebrate DNA methylation to normal
development [55].

DNA METHYLTRANSFERASES AND THE IMPORTANCE OF DNA METHYLATION IN
VERTEBRATES

Two advances reported in 1992 greatly aided DNA methylation research.
The first was a methodological breakthrough allowing much easier
analysis of DNA methylation at any DNA site of interest, namely
bisulfite-based genomic sequencing of m5C residues by direct
display of the methylated base [56], which also
gave rise to various valuable techniques for analysis of the
bisulfite-treated DNA by polymerase chain reaction (PCR) or PCR plus
restriction digestion. The basis for these methods is that methylation
of cytosine residues in DNA confers protection against deamination by
sequential bisulfite and mild alkali treatments [57, 58].

The second advance was the finding that insertional inactivation of the
most plentiful DNA methyltransferase in mammals was embryonic lethal in
mice [32]. Knockout of the other two main murine
DNA methyltransferase genes also leads to embryonic lethality or death
soon after birth [35]. Given the necessary
participation of all these genes (DNMT1, DNMT3A, and
DNMT3B) in setting and retaining DNA methylation patterns in
vertebrates, it would be easy for DNA methylation to have been lost
during vertebrate evolution if it did not play essential roles.
Although the enzymes encoded by these genes have other non-catalytic
functions, such as acting as repressors and recruiting histone
deacetylases [41-48, 59], these functions were apparently secondarily
acquired during evolution in the portions of the enzymes outside the
catalytic domain. In contrast to the rest of the sequence, the
C-terminal catalytic domain is shared by all prokaryotic and eukaryotic
DNA C-5 methyltransferase genes [60].

Evidence that the DNA methyltransferase function itself is necessary for
normal phenotypes comes from studies of a rare specific type of
chromosome instability syndrome called ICF (immunodeficiency,
centromeric region instability, facial anomalies) [61]. ICF is usually linked to mutations in both
alleles of DNMT3B in the C-terminal portion of the protein which
contains the catalytic domain. This domain is catalytically active even
when the rest of the protein is deleted [62]. The
mutations result in a minor decrease in overall DNA methylation, which
includes large decreases in satellite DNA methylation in several of the
satellite DNA sequences to which the chromosome abnormalities are
targeted [61, 63, 64]. Although DNMT3B has repressor activity that is
independent of its DNA methyltransferase activity, repression involves
portions of the protein that do not overlap the catalytic domain [48]. DNMT3B also forms a complex with DNMT1 and
DNMT3B and with small ubiquitin-like modifier 1, but these interactions
involve the N-terminus of DNMT3B [65, 66]. It is in one of ten motifs conserved among all
cytosine-C5 methyltransferases and present in the DNMT3B C-terminal
domain that many ICF-causing missense mutations are found, which
decrease DNMT3B's enzymatic activity [62]. These
findings suggest that it is the loss of DNA methyltransferase activity
and not some other function of the protein that is responsible for the
syndrome. The involvement of DNA hypomethylation in the phenotype of
ICF is supported at the cytogenetic level because the ICF-specific
rearrangements in mitogen-treated lymphocytes are the same in
frequency, spectrum, and chromosomal specificity as those we found in a
normal pro-B lymphoblastoid cell line treated with the DNA methylation
inhibitors 5-azacytidine or 5-azadeoxycytidine [67, 68].

DNA METHYLATION MODULATES EXPRESSION OF SOME VERTEBRATE GENES
DURING DEVELOPMENT AND FUNCTIONS IN GENE IMPRINTING AND X-CHROMOSOME
INACTIVATION

Enough thorough studies have now been reported to show that many tissue-
or development-specific changes in methylation at vertebrate promoters,
enhancers, or insulators regulate expression and are not simply
inconsequential by-products of expression differences [55].

First, there are mechanistic links that have been established between
DNA methylation and regulation of gene expression. DNA methylation can
affect histone modifications and chromatin structure, which, in turn,
can alter gene expression [27, 59, 69]. Increases in DNA
methylation can affect chromatin structure by increasing binding of
sequence-nonspecific methylated DNA binding proteins, which can recruit
histone deacetylases or other proteins to down-regulate transcription
[27].

Alternatively, increases or decreases in methylation of DNA sequences
can alter their interactions with sequence-specific DNA binding
proteins that bind less or more avidly to their CpG-containing
recognition sites when those sites are methylated [70-72]. During methylation of
hemimethylated DNA sequences in newly replicated DNA, a recruited DNA
methyltransferase can alter transcription by interacting itself with
histone deacetylases or other transcriptional repressors [59]. As described above, these interactions involve
methyltransferase domains other than the catalytic domain. Methylation
of special DNA elements called insulators can control long-distance
interactions of chromatin in cis by preventing insulator
activity and thereby allowing positive interactions of an enhancer on
one side of the insulator with a promoter on the other side [29, 73, 74].

Although CpG-rich mammalian promoters are often constitutively
hypomethylated, many show tissue-specific differences in DNA
methylation [75, 76]. There
is probably much overshooting in the control of establishment of
tissue-specific methylation patterns so that only a small percentage of
tissue-specific DNA methylation modulates gene expression. For various
genes with tissue-specifically methylated promoters, there is evidence
that changes in DNA methylation help regulate expression. For example,
the ALF gene, which specifies a germ cell-specific TFIIA
subunit, and a testis-specific lactate dehydrogenase gene are expressed
almost exclusively in testes or germinal epithelium [77, 78]. For both of these
genes, hypomethylation of the promoter in the expressed cells in
vivo and experiments in vitro implicate DNA methylation in
downregulation of expression or in maintaining the inactive state.
Other genes that display tissue-specific promoter hypomethylation and
expression and for which there is evidence that these in vivo
methylation differences are causally involved in regulation of the
gene's expression include the following: the myometrium-specific
oxytocin receptor gene, the liver-specific tyrosine aminotransferase
gene, the astrocyte- and astrocyte precursor-specific GFAP gene,
and the cytokine-encoding IFN-gamma gene [71, 79-86].

Another set of genes for which studies of animals, humans, and cultured
cells clearly demonstrate a role of DNA methylation in the regulation
of expression are those on the X chromosome. DNA methylation is not
necessary for establishing silencing of the inactive X chromosome
(Xi). However, it seems to be important in efficiently
protecting the one X chromosome that needs to stay active from
inactivation and for preventing reactivation of many silenced genes on
Xi once this inactivation is established [28, 87-89].

Differential DNA methylation is a critical signal for mammalian gene
imprinting, which gives the mono-allelic expression of imprinted genes
[29]. For most of the studied clusters of
imprinted genes, one allele is very highly methylated and the other
unmethylated or methylated at only a small percentage of CpGs in a 1-
to 5-kb CpG-rich region (differentially methylated region, DMR). The
gamete-specific differences in DMR methylation patterns, which are
usually at least partially retained during embryogenesis, appear to
generally be the primary imprinting mark.

Among the imprinted genes improperly expressed in
DNMT1-/- mouse embryos are H19, whose paternal
allele is normally silent, and the nearby IGF-2, whose maternal
allele is normally silent. In these mutant embryos, the paternal
H19 allele is abnormally activated, and the reciprocally
imprinted, paternal IGF-2 allele is abnormally silenced [29, 90]. Consistent with the
DNMT1 mutation acting through its effect on DNA methylation,
this mutation decreases methylation of the paternally imprinted DMR (an
insulator) between H19 and IGF-2 in mutant embryos.
Conversely, hypermethylation of this DMR on the paternal chromosome as
a result of engineered strong overexpression of DNMT1 in murine
embryonic stem cells is concomitant with bi-allelic expression of
IGF-2 [40]. In humans, inappropriate
methylation of this DMR in the paternal IGF2- and
H19-containing imprinted gene cluster due to cis-acting
imprinting mutations is found in certain patients with the
Beckwith-Wiedemann syndrome as well as in various cancers. Accompanying
this hypermethylation is bi-allelic IGF-2 expression, resulting
in abnormally high levels of its encoded mitogen and fetal growth
promoting protein. Both losses and gains of methylation in DMRs may
contribute to carcinogenesis via the resulting abnormal
expression of imprinted genes, which requires alteration of only one
allele for phenotypic changes [29, 91]. The demonstration of the involvement of Dnmt3L
[92] in indirectly contributing to
Dnmt3a-dependent DNA methylation necessary for spermatogenesis and
imprinting in mice further implicates DNA methylation in gene
imprinting [93]. However, unexpectedly it was
reported that human DNMT3L RNA is detectable only after birth [94]. This is another example of the complexity of DNA
methylation findings.

Controversial enigmas about vertebrate DNA methylation are still
arising. Several labs have found that the sperm-derived male pronucleus
in the zygote prior to nuclear fusion undergoes active and extensive
demethylation within hours of fertilization while the female pronucleus
undergoes passive demethylation (demethylation due to the absence of
methylation upon DNA replication) during the early cell divisions [95]. Similar findings were obtained for human, pig,
and rat embryos.

These results were interpreted as indicating a critical role for massive
DNA demethylation in early mammalian embryogenesis. Such zygote- and
early blastula-linked demethylation may be related to important
functions for gamete-specific DNA methylation patterns, imprinting, and
possible epigenetic problems associated with in vitro
fertilization. However, sheep embryos did not show evidence of
demethylation during the first post-fertilization cell cycle or after
the first mitosis [95]. Nonetheless in the
subsequent several cell divisions there is a considerable loss of DNA
methylation although it is not as widespread as in the mouse. Also,
Xenopus and zebra fish embryos do not display the extensive,
very early genomic demethylation seen in murine embryos.

Because the studies of zygote DNA methylation rely heavily on the use of
antibodies to m5C, the question of whether asymmetric
pronuclear methylation is an artifact reflecting antibody accessibility
has been raised even though there are controls in these studies [95]. Despite these interspecies differences in the
extent and timing of demethylation of DNA during early embryogenesis,
considerable demethylation has been observed in all studied mammals and
so its biological role needs to be carefully assessed.

DNA METHYLATION AND CANCER

Abnormal changes in DNA methylation postnatally are a major factor
contributing to oncogenesis. Both local increases in DNA methylation
and global decreases in genomic methylation are extremely common in
human cancer [91]. Abnormal DNA methylation in a
variety of human cancers relative to various normal somatic tissues was
first described in 1983 by our laboratory in collaboration with Charles
Gehrke using HPLC analysis of the total m5C content of DNA
digested to deoxynucleosides [9]. By Southern
blotting with a number of gene probes, Feinberg and Vogelstein in 1983
described frequent DNA hypomethylation in colon cancer [96, 97]. Both our study and
those of Feinberg and Vogelstein revealed decreases in DNA methylation
in cancer (DNA hypomethylation). Earlier reports of animal studies
indicated cancer-linked decreases in DNA methylation [5, 98-100]
as well as cancer-linked increases [101].

Hypermethylation of CpG-rich promoter regions specifically in human
cancers was first reported in 1986 for the calcitonin gene, which is
probably subject to de novo methylation during carcinogenesis,
not because it is a biologically important target, but rather, just
because it is caught in a wave of de novo methylation of certain
CpG-rich regions [102]. Subsequently, tumor
suppressor genes (TSGs) were shown to be frequently hypermethylated in
human cancer [103, 104].
Many later studies provide evidence that this hypermethylation is often
used to silence one or both TSG alleles during carcinogenesis in a wide
variety of tumors with tumor-specific profiles for which TSG promoters
are preferentially hypermethylated [105-108].

After the initial demonstrations that epigenetic inactivation of TSG is
a major mechanism for TSG silencing, research on DNA methylation
changes in cancer widely shifted to studying cancer-linked
hypermethylation, often ignoring the many reports [9, 109-117] of cancer-linked hypomethylation. Consistent
with that misleading oversimplification, many laboratories looked for
increases in DNA methyltransferase mRNA or protein to explain abnormal
DNA methylation in human malignancies [118-123].

Recently, it has become more widely appreciated that cancer-linked DNA
hypomethylation is just as prevalent as cancer-associated DNA
hypermethylation [91]. Early research on DNA
methylation and cancer implicated experimentally induced
hypomethylation in carcinogenesis or tumor progression [124-130]. Feeding rats and
mice methyl-deficient diets resulted in hepatocarcinogenesis, global
DNA undermethylation, and proto-oncogene demethylation although diet
effects other than DNA hypomethylation could contribute to tumor
formation [131-134].
However, these reports were largely overshadowed for a while by journal
articles implicating DNA hypermethylation causally in carcinogenesis
[135-138]. Recently
reported studies on transgenic mice subject to partial loss of DNA
methyltransferase activity [139-141] confirm the earlier studies implicating DNA
hypomethylation causally in oncogenesis.

It had been initially demonstrated by Vanyushin's group that the highly
repetitive fraction of vertebrate genomes is enriched in m5C
[5]. Studies from our laboratory, which were
subsequently confirmed by other laboratories, show that tandem DNA
repeats are frequently targeted for hypomethylation, sometimes to a
very large extent, in a variety of human cancers [142-149]. One of our studies
and a study from Itano et al. [149] revealed that
hypomethylation of tandemly repeated sequences is significantly
correlated with tumor progression and was an independent marker of poor
survival [117, 149], like
hypermethylation of gene regions [150]. It had
been proposed that DNA demethylation during carcinogenesis occurs prior
to de novo methylation, and that the functional significance of
this demethylation is just that it provokes TSG methylation.
Alternatively, it was hypothesized that DNA demethylation during cancer
formation is just a defensive attempt to counteract cancer-linked DNA
hypermethylation.

However, we have shown that global DNA hypomethylation and satellite DNA
hypomethylation in Wilms tumors and ovarian epithelial cancers are not
significantly associated with hypermethylation of most promoters of
TSGs although both DNA hypomethylation and hypermethylation are linked
to cancer [115, 151]. There
are a number of possible explanations for how DNA hypomethylation can
contribute to tumor formation and progression [91,
152], but the exact mechanisms are much less
clear than for TSG hypermethylation and its associated gene repression
[45, 153]. By whatever
mechanism cancer-linked DNA hypomethylation occurs and whatever its
most important biological targets, it should be more widely noted that
decreases in DNA methylation induced as part of a therapeutic regimen
might contribute to carcinogenesis [139, 140] or tumor progression [117, 149]. Therefore, DNA
hypomethylation therapies should be used only when less risky
alternatives are not available.

The reference list is necessarily incomplete, and the author apologizes
for the omission of other important contributions to the field of DNA
methylation.

This research was supported in part by NIH grant R01-CA81506.

This review is dedicated to Boris Vanyushin, a gracious leader in the
field of DNA methylation, whose research on animal DNA methylation was
the impetus for my laboratory's initial studies of the
tissue-specificity and then the cancer-specificity of human DNA
methylation.